Methods of downregulating translocated oncogene expression using bromodomain inhibitors

ABSTRACT

Disclosed herein are novel methods and compositions useful for inhibiting interaction between a bromodomain protein and an immunoglobulin (Ig) regulatory element. The methods and compositions are particularly useful for downregulating expression of an oncogene translocated with an Ig locus, as well as for treating a cancer (e.g., hematological malignancy) characterized by increased expression of an oncogene which is translocated with an Ig locus. Also disclosed herein are methods and assays for identifying agents that interfere with binding of bromodomain proteins to Ig regulatory elements, as well as methods and assays for identifying inhibitors of bromodomain

RELATED APPLICATION(S)

This application is a division of U.S. application Ser. No. 14/342,265,filed Sep. 2, 2014, (now U.S. Pat. No. 10,071,129) which is a nationalstage filing of PCT Application No. PCT/US2012/053173, filed Aug. 30,2012, which claims the benefit of U.S. Provisional Application No.61/529,165, filed on Aug. 30, 2011. The entire teachings of the aboveapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under NIH-1R01HG002668awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Oncogenes are a group of genes which, when activated, can cause cancer.Most normal cells undergo a programmed form of death (apoptosis);activated oncogenes can cause those cells that ought to die to surviveand proliferate instead. Most oncogenes require an additional step, suchas mutations in another gene or exposure to environmental factors, tocause cancer. One method of oncogenesis occurs by the process oftranslocation, in which a segment of the chromosome breaks off andattaches to another chromosome. If the dislocated chromosome contains anoncogene, it may be removed from its usual regulatory controls and becontinuously produced, thereby destabilizing the delicate balance of themechanisms of cell growth. Many leukemias and lymphomas are caused bytranslocations of oncogenes. Since the 1970s, dozens of oncogenes havebeen identified in human cancer.

MYC is an oncogene overexpressed in 30% of all human cancers, and inmany cancers it correlates with poor clinical outcome and increasedchance of relapse [1]. c-Myc links growth factor stimulation and cellproliferation [2, 3]. Mitogenic growth factor signaling induces MYCexpression. c-Myc, a basic helix-loop-helix leucine zipper transcriptionfactor, forms heterodimers with Max to positively regulate transcriptionof proliferation-associated genes which include genes involved inmetabolism, protein synthesis and cell-cycle [2, 3]. Thus, growth factorsignaling promotes cell proliferation through inducing c-Myc's functionin regulating transcription of proliferation-associated genes.

Myc is an attractive target for cancer therapy, as it is overexpressedin many human cancers that are difficult to treat and its expression islimited to proliferating cells. Studies in transgenic mouse modelssuggest that inactivation of MYC causes tumor regression through tumorcell differentiation or apoptosis [15-20]. In some models, such as themouse osteosarcoma model, brief MYC inactivation significantly improvedsurvival rates [19]. It has been suggested that the brief inactivationmight induce an epigenetic change in the tumor cells such thatreactivation would not necessarily cause tumor reformation. Thesestudies also revealed that a wide variety of tumors depend on c-Mycfunction to maintain their tumorigenic state [15-20].

In most cancers, MYC overexpression correlates with poor clinicaloutcome, aggressiveness and advanced stage of cancer [1, 21].Additionally, MYC overexpression correlates with an increasedproliferative capacity. Importantly, MYC overexpression correlates withlack of response to chemotherapy and increases the probability ofrelapse in many cancers [1]. Developing therapies targeting the c-Mycregulatory pathway would greatly improve the therapeutic options forthese aggressive, refractory malignancies.

SUMMARY OF THE INVENTION

The present invention relates to novel methods and compositions usefulfor inhibiting the interaction between a bromodomain protein and animmunoglobulin (Ig) regulatory element. The methods and compositions areparticularly useful for downregulating the expression of an oncogenewhich is translocated with an Ig locus, as well as for treating a cancer(e.g., hematological malignancy) characterized by increased expression(e.g., overexpression) of an oncogene which is translocated with an Iglocus. Also disclosed herein are methods and assays for identifyingagents that interfere with the binding of bromodomain proteins to Igregulatory elements, as well as methods and assays for identifyinginhibitors of bromodomain proteins.

Disclosed is a method of inhibiting interaction between a bromodomainprotein and an immunoglobulin (Ig) regulatory element comprisingcontacting said bromodomain protein with an effective amount of aninhibitor of said bromodomain protein.

Also disclosed is a method for downregulating expression of an oncogenetranslocated with an Ig locus comprising contacting said locus with aneffective amount of an inhibitor of a bromodomain protein.

Also disclosed is a method for treating a cancer comprising (a)determining the level of expression of an oncogene translocated with anIg locus in a sample obtained from an individual having or suspected ofhaving said cancer, wherein overexpression of said translocated oncogenein said sample in comparison with a control is indicative that saidindividual is an individual who would potentially benefit from treatmentwith an inhibitor of a bromodomain protein, and (b) administering aneffective amount of said inhibitor to said individual if saidtranslocated oncogene is overexpressed in said sample.

Also disclosed is a method for treating a cancer comprisingadministering an effective amount of an inhibitor of a bromodomainprotein to an individual exhibiting overexpression of an oncogenetranslocated with an Ig locus.

Also disclosed is the use of a bromodomain inhibitor for the treatmentof a cancer characterized by overexpression of an oncogene translocatedwith an Ig locus. In some embodiments use of the bromodomain inhibitorfor the treatment of cancer characterized by overexpression of anoncogene translocated with an Ig locus is with the proviso that saidoncogene is not c-Myc. In some embodiments use of the bromodomaininhibitor for the treatment of cancer characterized by overexpression ofan oncogene translocated with an Ig locus is with the proviso that saidcancer is not AML.

Also disclosed is a method for identifying an agent that interferes withbinding of a bromodomain protein to an Ig regulatory element comprising(a) contacting a suitably conditioned cell containing a target geneunder control of one or more Ig regulatory elements and a bromodomainprotein which binds to said one or more Ig regulatory elements andactivates expression of said target gene with a candidate agent, and (b)detecting expression of said target gene, wherein decreased expressionof said target gene in the presence of said candidate agent as comparedwith expression of said target gene in the absence of said candidateagent is indicative of said agent's ability to interfere with binding ofsaid bromodomain protein to said Ig regulatory element.

In some embodiments the method for identifying an agent that interfereswith binding of a bromodomain protein to an Ig regulatory elementfurther comprises the step of contacting said bromodomain proteinimmobilized onto a solid support with said candidate agent and a knowninhibitor of said bromodomain protein, wherein said candidate agent'sability to outcompete said known inhibitor for binding to saidbromodomain protein is indicative of said candidate agent's ability tointerfere with binding of said bromodomain protein to said Ig regulatoryelement.

In some embodiments the method for identifying an agent that interfereswith binding of a bromodomain protein to an Ig regulatory elementfurther comprises the step of comparing a level of bromodomain proteinoccupancy at said Ig regulatory element in the presence of saidcandidate agent to a control, wherein decreased bromodomain proteinoccupancy at said Ig regulatory element in the presence of saidcandidate agent as compared to in the absence of said candidate agent isindicative of bromodomain inhibitor activity of said candidate agent.

In some embodiments the method for identifying an agent that interfereswith binding of a bromodomain protein to an Ig regulatory elementfurther comprises the steps of (c) contacting said bromodomain proteinimmobilized onto a solid support with said candidate agent and a knowninhibitor of said bromodomain protein, wherein said candidate agent'sability to outcompete said known inhibitor for binding to saidbromodomain protein is indicative of said candidate agent's ability tointerfere with binding of said bromodomain protein to said Ig regulatoryelement, and (d) comparing a level of bromodomain protein occupancy atsaid Ig regulatory element in the presence of said candidate agent whichis able to outcompete said known inhibitor to a control, whereindecreased bromodomain protein occupancy at said Ig regulatory element inthe presence of said candidate agent as compared to in the absence ofsaid candidate agent is indicative of bromodomain inhibitor activity ofsaid candidate agent.

Disclosed also is a method for identifying an inhibitor of a bromodomainprotein comprising (a) contacting a cell line expressing an oncogenetranslocated with an Ig locus with a candidate agent, (b) measuring thelevel of bromodomain protein occupancy at a regulatory element of saidIg locus, and (c) comparing said level of bromodomain occupancy at saidregulatory element of said Ig locus to a control, wherein decreasedbromodomain protein occupancy at said regulatory element of said Iglocus in the presence of the candidate agent as compared to in theabsence of said candidate agent is indicative of bromodomain inhibitoryactivity of said candidate agent.

In some embodiments said Ig regulatory element is at an Ig locus on achromosome.

In some embodiments downregulating expression of said oncogene comprisesdecreasing transcription of said oncogene. In some embodimentsdownregulating expression of said oncogene comprises decreasing thelevel or activity of an expression product of said oncogene.

In some embodiments said oncogene is overexpressed as a result oftranslocation with said Ig locus. In some embodiments said oncogene isnot c-Myc. In some embodiments said oncogene is selected from the groupconsisting of Bcl2, Ccnd1, c-Maf, Pax5, Pim1, Bc16, Irf4, Il3, Lyt10,Bc13, and Malt1. In some embodiments said oncogene is c-Myc.

In some embodiments said Ig locus is selected from the group consistingof an IgH locus, an IgL locus, and an IgK locus.

In some embodiments said inhibitor interferes with binding of saidbromodomain protein to an IgH regulatory element. In some embodimentssaid inhibitor interferes with binding of said bromodomain protein to atranscriptional start site of said oncogene. In some embodiments saidinhibitor interferes with binding of said bromodomain protein to atranscriptional start site of an oncogene translocated with said Iglocus. In some embodiments said inhibitor interferes with acetyl-lysinerecognition by a central hydrophobic cavity of said bromodomain protein.In some embodiments said inhibitor interferes with acetyl-lysineanchoring by a hydrogen bond of an asparagine residue of saidbromodomain protein. In some embodiments said inhibitor inhibits bindingof said bromodomain protein to an enhancer of said Ig locus. In someembodiments said inhibitor is selected from the group consisting of anantisense oligonucleotide, an aptamer, an intrabody, an oligopeptide, aribozyme, an siRNA, a shRNA, and a small molecule.

In some embodiments said Ig regulatory element is an Ig enhancer.

In some embodiments said enhancer is an IgH enhancer. In someembodiments said enhancer is selected from the group consisting of E1,E2, E3 and E4.

In some embodiments said bromodomain protein is a BET bromodomainprotein. In some embodiments said bromodomain protein is selected fromthe group consisting of Brd2, Brd3, Brd4, and BrdT.

In certain embodiments said oncogene is c-Myc, said Ig locus is an IgHlocus, and said bromodomain protein is Brd4.

In some aspects said cancer is not AML. In some aspects said cancer is ahematological malignancy.

In some aspects said hematological malignancy is a leukemia, lymphoma ormyeloma selected from the group consisting of acute lymphoblasticlymphoma (ALL), Burkitt's lymphoma, chronic lymphoid leukemia (CLL),diffuse large cell lymphoma, extranodal lymphoma, follicular lymphoma,lymphoplasmacytoid lymphoma, mantle zone lymphoma, monoclonal gammopathyof undetermined significance (MGUS), multiple myeloma, andmucosa-associated lymphatic tissue (MALT)-type lymphoma. In some aspectssaid hematological malignancy is acute myeloid leukemia (AML).

In certain embodiments said oncogene is bcl-1, said Ig locus is an IgHlocus, and said cancer is mantle zone lymphoma. In certain embodimentssaid oncogene is bcl-2, said Ig locus is an IgH locus, and said canceris follicular lymphoma. In certain embodiments said oncogene is bcl3,said Ig locus is an IgH locus, and said cancer is B-cell chroniclymphocytic leukemia. In certain embodiments said oncogene is bcl-6,said Ig locus is an IgH locus, and said cancer is diffuse large celllymphoma. In certain embodiments said oncogene is bcl-9, said Ig locusis an IgH locus, and said cancer is acute lymphoblastic lymphoma. Incertain embodiments said oncogene is bcl-10 and said cancer ismucosa-associated lymphatic tissue (MALT)-type lymphoma. In certainembodiments said oncogene is c-maf, said Ig locus is an IgH locus, andsaid cancer is multiple myleloma. In certain embodiments said oncogeneis c-myc, said Ig locus is an IgH locus, and said cancer is Burkitt'slymphoma. In certain embodiments said oncogene is c-myc, said Ig locusis an IgH locus, and said cancer is multiple myeloma. In certainembodiments said oncogene is FGFR3, said Ig locus is an IgH locus, andsaid cancer is multiple myeloma. In certain embodiments said oncogene isLyt-10, said Ig locus is an IgH locus, and said cancer is diffuse largecell lymphoma. In certain embodiments said oncogene is MUC1, said Iglocus is an IgH locus, and said cancer is extranodal lymphoma. Incertain embodiments said oncogene is MUM1/IRF4, said Ig locus is an IgHlocus, and said cancer is multiple myeloma. In certain embodiments saidoncogene is Pax-5, said Ig locus is an IgH locus, and said cancer islymphoplasmacytoid lymphoma.

In some aspects said inhibitor is an inhibitor of a BET bromodomainprotein. In some aspects said inhibitor is an inhibitor of Brd4 protein.In some aspects said inhibitor is small molecule JQ1. In some aspectssaid inhibitor is administered with a pharmaceutically acceptablecarrier. In some aspects said inhibitor is co-administered with at leastone chemotherapeutic agent.

In some aspects said candidate agent interferes with acetyl-lysinerecognition by a central hydrophobic cavity of said bromodomain proteinor interferes with acetyl-lysine anchoring by a hydrogen bond of anasparagine residue of said bromodomain protein.

In some aspects said agent or candidate agent is selected from the groupconsisting of an antisense oligonucleotide, an aptamer, an intrabody, anoligopeptide, a ribozyme, an siRNA, a shRNA, and a small molecule.

In some aspects said Ig regulatory element is an IgH regulatory element.

In some aspects said target gene is a reporter gene. In some aspectssaid target gene is fused to a protein tag. In some aspects said proteintag is selected from the group consisting of a fluorescent peptide and apoly His tag. In some aspects said target gene is an oncogene which istranslocated with an Ig locus. In some aspects said oncogene is notc-Myc.

In certain embodiments said bromodomain protein is Brd4, said oncogeneis c-Myc, and said Ig regulatory element is an IgH enhancer.

In some embodiments said control bromodomain occupancy level isdetermined by contacting said cell line with a known bromodomain proteininhibitor, and measuring a level of bromodomain occupancy at saidregulatory element of said Ig locus.

In some embodiments said known bromodomain protein inhibitor is smallmolecule JQ1.

In some embodiments said cell line is a multiple myeloma cell line. Insome embodiments said Ig regulatory element is an IgH enhancer.

In some embodiments said step of measuring said level of bromodomainprotein occupancy at said regulatory element of said Ig locus isperformed using bromodomain protein ChIP-PCR analysis. In otherembodiments, said step of measuring said level of bromodomain proteinoccupancy at said regulatory element of said Ig locus can be performedusing sequencing techniques. In still other embodiments, said step ofmeasuring said level of bromodomain protein occupancy at said regulatoryelement of said Ig locus can be performed using microarrays (e.g., a DNAmicroarray).

In some embodiments said ChIP-PCR analysis is performed usingoligonucleotide primers complementary to at least a portion of asequence of said regulatory element.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 10^(th) ed. (2006)or 11th edition (July 2009). Non-limiting information regarding genesand genetic disorders is found in McKusick, V. A.: Mendelian Inheritancein Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIM™. McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates that Brd4 binds the IGH regulator elements in IGH-MYCtranslocated multiple myeloma. Brd4 occupancy in MM1S cells at MYCenhancer regions, MYC TSS and IGH enhancer regions using two separateantibodies recognizing Brd4 (Bethyl).

FIG. 2 shows that Brd4 binds the IGH regulator elements in IGH-MYCtranslocated multiple myeloma. Brd4 occupancy in MM1S cells at MYCenhancer regions, MYC TSS and IGH enhancer regions using two separateantibodies recognizing Brd4 (Sigma).

DETAILED DESCRIPTION OF THE INVENTION

Described herein are novel methods, assays and compositions for theinhibition of bromodomain proteins as a mechanism to downregulateexpression of oncogenes translocated with immunoglobulin (Ig) loci onchromosomes, and methods of identifying inhibitors of bromodomainproteins. In particular, described herein is the discovery thatbromodomain proteins bind to the immunoglobulin (Ig) regulatory elementsof oncogenes translocated with Ig loci in a way that induces oncogeneoverexpression, and that bromodomain protein inhibitors interfere withbromodomain protein binding to such Ig regulatory elements todownregulate expression of such oncogenes translocated with Ig loci. Themethods, assays and compositions disclosed herein also provide novelstrategies for identifying bromodomain protein inhibitors that areuseful for interfering with bromodomain protein binding to Ig regulatoryelements.

Disclosed herein are methods for inhibiting the interaction between abromodomain protein and an immunoglobulin (Ig) regulatory element, suchmethods comprising contacting said bromodomain protein with an effectiveamount of an inhibitor of said bromodomain protein. Bromodomain proteinsrefer to human proteins containing at least one bromodomain whichmediates molecular recognition of acetyl-lysine in the aceylation oflysine residues on the tails of histones (which are typically correlatedwith an accessible chromatin structure and transcriptional activation)and non-histone proteins. Bromodomains generally share a conserved foldcharacterized by a left-handed bundle of four α-helices which are linkedby regions of diverse loops that confer substrate specificity. Themajority of bromodomains contain a central hydrophobic cavity thatrecognizes acetyl-lysine on the tails of histones and anchorsacetyl-lysine via a hydrogen bond to a conserved asparagine residue inthe binding pocket of the bromodomain. As used herein “bromodomain” or“bromodomain protein” refers to a polypeptide, whether wild-type ormutant, natural or synthetic, truncated or complete, or a variantthereof that possesses the minimum amino acid sequence sufficient for afunctional bromodomain capable of mediating molecular recognition ofacetyl-lysine of acetylated lysine residues on the tails of histones.Useful bromodomain proteins can include, for example, fusion proteinscomprising a bromodomain and an additional portion having desiredfunctionality (e.g., a reporter portion).

As used herein “contacting the cell” and the like, refers to any meansof introducing an agent (e.g., nucleic acid, oligopeptide, ribozyme,intrabody, small molecule, etc.) into a target cell in vitro or in vivo,including by chemical and physical means, whether directly or indirectlyor whether the agent physically contacts the cell directly or isintroduced into an environment (e.g., culture medium) in which the cellis present or to which the cell is added. Contacting also is intended toencompass methods of exposing a cell, delivering to a cell, or ‘loading’a cell with an agent by viral or non-viral vectors, and wherein suchagent is bioactive upon delivery. The method of delivery will be chosenfor the particular agent and use (e.g., cancer being treated).Parameters that affect delivery, as is known in the art, can include,inter alia, the cell type affected (e.g., tumor), and cellular location.In some embodiments, “contacting” includes administering the agent to anindividual. In some embodiments, “contacting” refers to exposing a cellor an environment in which the cell is located to one or morebromodomain inhibitors of the present invention. In some embodiments,“contacting” refers to exposing a cell or an environment in which thecell is located to one or more candidate agents of the presentinvention.

As used herein an “effective amount” or “effective dose” of a compoundor other agent (or composition containing such compound or agent) refersto the amount sufficient to achieve a desired biological and/orpharmacological effect, e.g., when delivered to a cell or organismaccording to a selected administration form, route, and/or schedule. Aswill be appreciated by those of skill in the art, the absolute amount ofa particular compound, agent, or composition that is effective may varydepending on such factors as the desired biological or pharmacologicalendpoint, the agent to be delivered, the target tissue, etc. Those ofskill in the art will further understand that an “effective amount” maybe contacted with cells or administered in a single dose, or the desiredeffect may be achieved by use of multiple doses. An effective amount ofa composition may be an amount sufficient to reduce the severity of orprevent one or more symptoms or signs of a disorder.

Also disclosed herein are methods for downregulating expression of anoncogene translocated with an Ig locus, such methods comprisingcontacting said locus with an effective amount of an inhibitor of abromodomain protein. As used herein, “downregulating expression” refersto a reduction (measurable or observable) in the expression of atranscription or translation product of the oncogene in a target cell orcell population, or target tissue. In some embodiments, downregulatingexpression of the oncogene refers to decreasing the transcription of theoncogene. In some embodiments, downregulating expression of saidoncogene comprises decreasing the level or activity of an expressionproduct of said oncogene. As used herein “level” refers to a measure ofthe amount of or a concentration of a transcription product, forinstance an mRNA, or a translation product, for instance a protein orpolypeptide. As used herein “activity” refers to a measure of theability of a transcription product or a translation product to produce abiological effect.

In some embodiments, the oncogene is overexpressed as a result oftranslocation with said Ig locus. As used herein “overexpression,”“overexpressed” or “overexpression of an oncogene” are usedinterchangeably to refer to a level of expression (e.g., an amount ofmRNA or protein produced, e.g., oncogene expression) or the amount ofactivity (e.g., bromodomain protein binding acetylated lysine residues)that is generally at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold ormore higher than a reference or normal level and/or activity. However,modestly increased levels and/or activity, such as about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9-fold higher levels and/or activity than areference or normal level or activity of an oncogene (e.g., an oncogenetranslocated with an Ig locus) or its expression are also encompassed bythis phrase.

As described herein, any oncogene which is translocated with an Ig locus(e.g., an Ig heavy or Ig light locus) may be beneficially targeted bythe methods disclosed herein. Such methods of inhibiting ordownregulating the expression of translocated oncogene are useful fortreating disorders characterized by expression, increased expression, oroverexpression of such translocated oncogenes. Briefly, and withoutwishing to be bound by theory, it is believed that oncogenetranslocation with Ig loci places the translocated oncogene undercontrol of the Ig regulatory elements, which can lead to increasedexpression or overexpression because such Ig loci are highly expressedin B- and T-cells. This causes increased expression or overexpression ofthe translocated oncogene and uncontrolled proliferation of the cell.

By way of non-limiting example, the oncogene can be one or more of Bcl2,Bc13, Bc16, Bc19, Bc10, Ccnd1, c-Maf, c-myc, Pax5, Pim1, Irf4, Il3,Lyt10, and Malt1. In certain embodiments the oncogene is not c-myc.

In one example, expression of oncogene c-myc translocated with an Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor as described herein. In oneembodiment, overexpressed oncogene c-Myc translocated with an IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor as described herein.

As another example, expression of oncogene Bcl2 translocated with an Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor as described herein. In oneembodiment, overexpressed oncogene Bcl2 translocated with an IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor as described herein.

In one embodiment, expression of oncogene Bc13 translocated with an Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor according to methods describedherein. For example, overexpressed oncogene Bc13 translocated with anIgH locus can be inhibited or downregulated by contacting the IgH locuswith an effective amount of a bromodomain inhibitor.

In another embodiment, expression of oncogene Bc16 translocated with anIg locus can be downregulated by contacting the Ig locus with aneffective amount of a bromodomain protein inhibitor as described herein.In one embodiment, overexpressed oncogene Bc16 translocated with an IgHlocus can be inhibited or downregulated by contacting the IgH locus withan effective amount of a bromodomain inhibitor. In another embodiment,overexpressed oncogene Bc16 translocated with an IgL locus can beinhibited or downregulated by contacting the IgL locus with an effectiveamount of a bromodomain inhibitor as described herein.

In a further embodiment, expression of oncogene Bc19 translocated withan Ig locus can be downregulated by contacting the Ig locus with aneffective amount of a bromodomain protein inhibitor. For example,overexpressed oncogene Bc19 translocated with an IgH locus can beinhibited or downregulated by contacting the IgH locus with an effectiveamount of a bromodomain inhibitor.

In another embodiment, expression of oncogene Bc110 translocated with anIg locus (e.g., an IgH locus) can be downregulated by contacting the Iglocus with an effective amount of a bromodomain protein inhibitor. Inone embodiment, overexpressed oncogene Bc110 translocated with an IgHlocus can be inhibited or downregulated by contacting the IgH locus withan effective amount of a bromodomain inhibitor as described herein.

In one embodiment, expression of oncogene Ccnd1 translocated with an Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor according to methods of theinvention. In an embodiment, overexpressed oncogene Ccnd1 translocatedwith an IgH locus can be inhibited or downregulated by contacting theIgH locus with an effective amount of a bromodomain inhibitor.

In one embodiment, expression of oncogene c-Maf translocated with an Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor. In one embodiment,overexpressed oncogene c-Maf translocated with an IgH locus can beinhibited or downregulated by contacting the IgH locus with an effectiveamount of a bromodomain inhibitor.

In another example, expression of oncogene Pax5 translocated with Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor of the present invention. Inan embodiment, overexpressed oncogene Pax5 translocated with IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, expression of oncogene Pim1 translocated with Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor of the present invention. Inan embodiment, overexpressed oncogene Pim1 translocated with IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, expression of oncogene Irf4 translocated with Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor of the present invention. Inan embodiment, overexpressed oncogene Irf4 translocated with IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, expression of oncogene Il3 translocated with Ig locuscan be downregulated by contacting the Ig locus with an effective amountof a bromodomain protein inhibitor of the present invention. In anembodiment, overexpressed oncogene Il3 translocated with IgH locus canbe inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, expression of oncogene Lyt10 translocated with Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor of the present invention. Inan embodiment, overexpressed oncogene Lyt10 translocated with IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, expression of oncogene Malt1 translocated with Iglocus can be downregulated by contacting the Ig locus with an effectiveamount of a bromodomain protein inhibitor of the present invention. Inan embodiment, overexpressed oncogene Malt1 translocated with IgH locuscan be inhibited or downregulated by contacting the IgH locus with aneffective amount of a bromodomain inhibitor of the present invention.

In one embodiment, the oncogene is c-Myc. For example, expression ofoncogene c-Myc translocated with Ig locus can be downregulated bycontacting the Ig locus with an effective amount of a bromodomainprotein inhibitor of the present invention. In another example,overexpressed oncogene c-Myc translocated with IgH locus can beinhibited or downregulated by contacting the IgH locus with an effectiveamount of a bromodomain inhibitor of the present invention. In anembodiment, overexpressed oncogene c-Myc translocated with IgL locus canbe inhibited or downregulated by a contacting the IgL locus with aneffective amount of a bromodomain inhibitor of the present invention.

It should be appreciated by those skilled in the art that any Ig locuswith which an oncogene can be translocated may result in overexpressionof the translocated oncogene. In such instances, the bromodomain proteininhibitors and methods for inhibiting or downregulating translocatedoncogene expression disclosed herein may be advantageously employed. Incertain embodiments, the Ig locus is selected from the group consistingof an IgH locus, an IgL locus, and an IgK locus.

In one embodiment, the Ig locus in which a translocation occurs is theIgH locus. The human IgH locus is located on the chromosome 14 at band14q32.33, adjacent to the telomeric extremity of the long arm. The humanIgH locus at 14q32.33 spans 1250 kilobases (kb). It contains 123 to 129IgHV genes, depending on the haplotypes, 27 IgHD segments which belongto 7 subgroups, 9 IgHJ segments, and 11 IgHC genes. The IgH locusencodes immunoglobulin heavy chains which result from the rearrangementof the IgHV, IgHD and IgHJ genes. Translocations involving the IgH locusmay result from errors of recombinase enzyme complex (RAG1, RAG2, etc.),Ig and T-cell receptor V-J or V-D-J rearrangements, or switch enzymes.

In one embodiment, the Ig locus in which a translocation occurs is theIgL locus. The human IgL locus is located on the chromosome 22 on thelong arm at band 22q11.2.

The human IgL locus spans approximately 1050 kb and contains 29 to 32functional IgLV genes which belong to 10 subgroups, 4 to 5 IgLJ, 4 to 5IgLC functional genes in the 7-IgLC haplotype. The IgL locus encodesimmunoglobulin lambda chains which result from the rearrangement of theIgLV, IgLJ genes with a deleted intermediate DNA region to produce arearranged IgLV-J gene which is transcribed with a IgLC gene andtranslated to produce the Ig lambda chain. Translocations involving theIgH locus may result from errors of recombinase enzyme complex (RAG1,RAG2, etc.), or T-cell receptor V-J or V-D-J rearrangements.

In one embodiment, the Ig locus in which a translocation occurs is theIgK locus. The human IgK locus is located on the chromosome 2 on theshort arm at band 2p11.2.

The human IgK locus contains genes for the kappa light chains ofimmunoglobulins which are encoded by IgKV, IgKJ, and IgKC genes whichundergo VDJ recombination events which may result in translocations withoncogenes.

In certain embodiments, the present invention contemplates methods ofinhibiting the function, level, or activity of a bromodomain domainprotein using one or more bromodomain inhibitor of the presentinvention. As used herein “bromodomain inhibitor”, “inhibitor ofbromodomain protein” and “bromodomain protein inhibitor” refer toinhibitors of bromodomain proteins, and in particular inhibitors whichinterfere with the ability of bromodomain proteins to interact withacetylated lysine residues residing on the tails of histones, includingagents that inhibit the level and/or activity of bromodomain protein, oragents that downregulate transcription of a gene encoding a bromodomainprotein. Those skilled in the art will appreciate that interference withthe ability of a bromodomain protein to interact with acetylated lysineson histone tails may refer to preventing the bromodomain protein fromrecognizing or binding to the acetyl-lysine, competing withacetyl-lysine for hydrogen bonding to a conserved asparagine residuepresent in the central hydrophobic cavity or binding pocket of thebromodomain so as to displace the bromodomain protein from thechromatin, disrupting the hydrogen bond between acetyl-lysine and theconserved asparagine residue so as to displace the bromodomain proteinfrom the acetyl-lysine motif, or any other means by which molecularrecognition between acetyl-lysine and the bromodomain is disruptedpreventing the bromodomain protein from contributing to the formation ofa functional transcription factor complex and thereby modulating geneexpression. In some embodiments, bromodomain protein inhibitors includemolecules that bind directly to a functional region of a bromodomainprotein. In certain embodiments, the bromodomain inhibitor interfereswith binding of a bromodomain protein to an Ig regulatory element. Insome embodiments, the bromodomain inhibitor interferes with binding of abromodomain protein to an IgH regulatory element. In some embodiments,the bromodomain inhibitor interferes with binding of said bromodomainprotein to a transcriptional start site of an oncogene. In certainembodiments, the bromodomain inhibitor interferes with binding of abromodomain protein to a transcriptional start site of an oncogenetranslocated with an Ig locus. In some embodiments, the bromodomaininhibitor interferes with acetyl-lysine recognition by a centralhydrophobic cavity of said bromodomain protein. In some embodiments, thebromodomain inhibitor interferes with acetyl-lysine anchoring by ahydrogen bond of an asparagines residue of a bromodomain protein. Insuch instances, the asparagines residue is a conserved residue whichresides in a binding pocket or central hydrophobic pocket of thebromodomain protein.

In some embodiments, the present invention contemplates bromodomaininhibitors that interfere with the interaction between a bromodomainprotein and an Ig regulatory element. It should be appreciated by thoseskilled in the art that the Ig regulatory element may be any regulatoryelement that is part of a functional Ig gene or Ig locus translocatedwith an oncogene. In some embodiments, the Ig regulatory element is anIg enhancer. In such instances, the bromodomain inhibitor inhibitsbinding of the bromodomain protein to an enhancer of the Ig locus. Insome embodiments, the enhancer is an IgH enhancer. In some embodiments,the enhancer is selected from the group consisting of E1, E2, E3 and E4.Ig enhancers E1, E2, E3 and E4 are described in further detail inExample 1 below. In some embodiments, the enhancer is a regulatoryelement having a nucleic acid sequence substantially complementary to orsimilar to an oligonucleotide sequence listed in Table 1. In someembodiments, the enhancer is a putative Ig enhancer. In otherembodiments, the regulatory element comprises a nucleotide sequenceadjacent to an Ig enhancer, a binding site within such nucleotidesequence, or a transcription factor which binds to such sequence.

It should be appreciated that any method or agent capable of inhibitinga bromodomain protein now known, or later discovered, can be used inaccordance with the methods of the present invention to inhibit abromodomain protein or to downregulate expression of an oncogenetranslocated with an Ig locus. Examples of such methods or agents knownto those skilled in the art include, but are not limited to antisenseoligonucleotides, oligopeptides, interfering RNA e.g., small interferingRNA (siRNA), small hairpin RNA (shRNA), aptamers, ribozymes, smallmolecule inhibitors, or intrabodies, and combinations thereof.

In certain embodiments, the bromodomain protein is a BET bromodomainprotein. In such instances the bromodomain protein may include Brd2,Brd3, and Brd4. In one embodiment, the bromodomain protein is BrdT.

In one exemplary embodiment, bromodomain protein Brd4 binds to an IgHregulatory element that induces overexpression of oncogene c-Myctranslocated with IgH locus. In such embodiment, contacting said c-Myconcogene translocated with IgH locus with an effective amount of abromodomain protein inhibitor of the present invention (e.g., a BETbromodomain inhibitor, e.g., a Brd4 inhibitor) may be advantageouslyperformed to downregulate the expression of c-Myc translocated with IgH.

In another aspect, a method for treating a cancer is disclosed herein,such method comprising (a) determining the level of expression of anoncogene translocated with an Ig locus in a sample obtained from anindividual having or suspected of having said cancer, whereinoverexpression of said translocated oncogene in said sample incomparison with a control is indicative that said individual is anindividual who would potentially benefit from treatment with aninhibitor of a bromodomain protein; and (b) administering an effectiveamount of said inhibitor to said individual if said translocatedoncogene is overexpressed in said sample.

In yet another aspect, a method for treating a cancer comprisesadministering an effective amount of an inhibitor of a bromodomainprotein to an individual exhibiting overexpression of an oncogenetranslocated with an Ig locus. In one embodiment, the cancer is notacute myeloid leukemia.

As used herein “treat”, “treating” and similar terms refer to providingmedical and/or surgical management of a subject. Treatment can include,but is not limited to, administering a compound or composition (e.g., apharmaceutical composition or a composition comprising appropriate cellsin the case of cell-based therapy) to a subject. Treatment is typicallyundertaken in an effort to alter the course of a disorder (which term isused to refer to a disease, syndrome, or abnormal condition) orundesirable or harmful condition in a manner beneficial to the subject.The effect of treatment can generally include reversing, alleviating,reducing severity of, delaying the onset of, curing, inhibiting theprogression of, and/or reducing the likelihood of occurrence orreoccurrence of the disorder or condition, or one or more symptoms ormanifestations of such disorder or condition. A composition can beadministered to a subject who has developed a disorder or is at risk ofdeveloping a disorder (e.g., cancer, e.g., a hematological malignancy).A composition can be administered prophylactically, i.e., beforedevelopment of any symptom or manifestation of a disorder. Typically inthis case the subject will be at increased risk of developing thedisorder relative to a member of the general population. For example, acomposition can be administered to a subject with a risk factor, e.g., atranslocation of an oncogene with an Ig locus, wherein the risk factoris associated with increased likelihood of developing the disorder butbefore the subject has developed symptoms or manifestations of thedisorder. “Preventing” can refer to administering a composition to asubject who has not developed a disorder, so as to reduce the likelihoodthat the disorder will occur or so as to reduce the severity of thedisorder should it occur. The subject may be identified (e.g., diagnosedby a medical practitioner) as having, suspected of having, or being atrisk of developing the disorder (e.g., at increased risk relative tomany most other members of the population or as having a risk factorthat increases likelihood of developing the disorder).

As used herein determining or detecting the level of expression of agene or oncogene (e.g., an oncogene translocated with Ig locus) refersto any method that can be used to detect the level of expression of atarget gene, including mRNA levels and/or the activity or level of aprotein encoded by that gene. Methods for determining the level ofexpression of a particular gene are well known in the art. Any suchmethod now known or later developed can be used to determine or detectthe levels of gene or oncogene expression in accordance with the methodsdisclosed herein. Examples of such suitable methods include RT-PCR,real-time PCR, Northern blotting, Western blotting, in situhybridization, oligonucleotide arrays (e.g., microarray) or chips, toname more than a few. Such methods can be used to detect gene expressionor overexpression of a gene (e.g., an oncogene translocated with an Iglocus)

As used herein “sample” or “biological sample” are used interchangeablyto refer to a sample of biological tissue or fluid that contains nucleicacids or polypeptides, e.g., an oncogene translocated with an Ig locus.Such samples may include isolated tissues or sections of tissues such asbiopsy or autopsy samples, frozen sections taken for histologicalpurposes, blood, plasma, serum. A biological sample may also refer totransformed cell cultures derived from patient tissues. Those skilled inthe art will appreciate that any methods now known, or later developed,for isolating, extracting, or obtaining a sample with sufficientmeasurable quanitites of nucleic acids or polypeptides can be used inaccordance with the methods disclosed herein.

As used herein “administering” a bromodomain protein inhibitor orcomposition thereof may be performed according to any suitable methodnow known, or later developed, such as intravenous administration, e.g.,as a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. Other therapeutic regimens may be combined with theadministration of the bromodomain protein inhibitors. The combinedadministration may include co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein there may be a time period whileboth (or all) active agents simultaneously exert their biologicalactivities. Such combined therapy may generate a synergistic therapeuticeffect.

For the treatment of a cancer, the dosage and mode of administrationwill be chosen by the physician according to known criteria. Atherapeutically effective dose of the bromodomain protein inhibitoralone, or linked to a cancer therapeutic agent, is the amount effectivefor inhibiting the binding of the bromodomain protein to an Igregulatory element thereby downregulating expression of an oncogenetranslocated with an Ig locus. The dosage should not cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand route of administration, or whether other drugs are included in theregimen, and can be determined by one of skill in the art. Theappropriate dosage of the bromodomain inhibitor will also depend on thetype of cancer (e.g., leukemia, myeloma, lymphoma, etc.) to be treatedand the severity and course of the disease. The bromodomain inhibitormay be appropriately administered to the patient at one time or over aseries of treatments.

In some embodiments, the cancer to be treated according to the methodsdescribed herein is a hematological malignancy. As used herein, “bloodcancer”, “hematological malignancy”, “hematological cancer”,“hematopoietic malignancy” and “hematopoietic cancer” are usedinterchangeably to refer to diseases affecting the blood, bone marrow,and lymph nodes. In some embodiments, a hematological malignancy refersto a leukemia, a lymphoma or a myeloma and specific disease typesthereof. Examples of such hematological malignancies include acutelymphoblastic lymphoma (ALL), Burkitt's lymphoma, chronic lymphoidleukemia (CLL), diffuse large cell lymphoma, extranodal lymphoma,follicular lymphoma, lymphoplasmacytoid lymphoma, mantle zone lymphoma,monoclonal gammopathy of undetermined significance (MGUS), multiplemyeloma, and mucosa-associated lymphatic tissue (MALT)-type lymphoma.Other examples of hematological malignancies include Waldenstrom'smacroglobulinemia, heavy chain disease, chronic myelogenous leukemia(CML), hairy cell leukemia, promyelocytic leukemia, myelomonocyticleukemia, monocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma(small-cell type, large-cell type, and mixed-cell type). In oneembodiment, the hematological malignancy is acute myeloid leukemia(AML).

The methods of treating cancer disclosed herein relate to a variety ofcancers characterized by abnormal expression (e.g., overexpression) ofoncogenes translocated with Ig loci. It should be appreciated that anyoncogene which is translocated with an Ig locus may exhibit abnormalexpression (e.g., overexpression), and thus can be targeted fordownregulation or inhibition as a therapeutic strategy for treatingcancer with the bromodomain inhibitors disclosed herein or identified inaccordance with the methods disclosed herein. In one embodiment, theoncogene is not c-Myc.

In one embodiment, the oncogene is bcl-1, the Ig locus is an IgH locus,and the cancer is mantle zone lymphoma. For example, patients having orsuspected of having mantle zone lymphoma may exhibit oncogene bcl-2translocated with an IgH locus. In some instances, oncogene bcl-2translocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene bcl-2 translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients in which oncogene bcl-2translocated with an IgH locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain proteininhibitor.

In one embodiment, the oncogene is bcl-2, the Ig locus is an IgH locus,and the cancer is follicular lymphoma. For example, patients having orsuspected of having follicular lymphoma may exhibit oncogene bcl-2translocated with an IgH locus. In some instances, oncogene bcl-2translocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene bcl-2 translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients in which oncogene bcl-2translocated with an IgH locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain proteininhibitor.

In one embodiment, the oncogene is bcl-3, the Ig locus is an IgH locus,and the cancer is B-cell chronic lymphocytic leukemia. For example,patients having or suspected of having B-cell chronic lymphocyticleukemia may exhibit oncogene bcl-3 translocated with an IgH locus. Insome instances, oncogene bcl-3 translocated with an IgH locus may beoverexpressed in such patients. The expression level of oncogene bcl-3translocated with an IgH locus may be detected in such patients usingknown methods of measuring or detecting gene expression. Those patientshaving or suspected of having B-cell chronic lymphocytic leukemia inwhich oncogene bcl-3 translocated with an IgH locus is overexpressed maybenefit from administration of an effective amount of a bromodomainprotein inhibitor in accordance with the methods disclosed herein. Inone embodiment, the bcl-3 oncogene translocation is a [t(14;19)]chromosomal translocation.

In one embodiment, the oncogene is bcl-6, the Ig locus is an IgH locus,and the cancer is diffuse large cell lymphoma. For example, patientshaving or suspected of having diffuse large cell lymphoma may exhibitoncogene bcl-6 translocated with an IgH locus. In some instances,oncogene bcl-6 translocated with an IgH locus may be overexpressed insuch patients. The expression level of oncogene bcl-6 translocated withan IgH locus may be detected in such patients using known methods ofmeasuring or detecting gene expression. Those patients having orsuspected of having diffuse large cell lymphoma in which oncogene bcl-6translocated with an IgH locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain protein inhibitorin accordance with the methods disclosed herein. In an embodiment, thebcl-6 oncogene translocation is a [t(3;14)] chromosomal translocation.

In one embodiment, the oncogene is bcl-9, the Ig locus is an IgH locus,and the cancer is acute lymphoblastic lymphoma. For example, patientshaving or suspected of having acute lymphoblastic lymphoma may exhibitoncogene bcl-9 translocated with an IgH locus. In some instances,oncogene bcl-9 translocated with an IgH locus may be overexpressed insuch patients. The expression level of oncogene bcl-9 translocated withan IgH locus may be detected in such patients using known methods ofmeasuring or detecting gene expression. Those patients having orsuspected of having acute lymphoblastic lymphoma in which oncogene bcl-9translocated with an IgH locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain protein inhibitorin accordance with the methods disclosed herein.

In one embodiment, the oncogene is bcl-10 and the cancer ismucosa-associated lymphatic tissue (MALT)-type lymphoma. For example,patients having or suspected of having mucosa-associated lymphatictissue (MALT)-type lymphoma may exhibit oncogene bcl-10 translocatedwith an IgH locus. In some instances, oncogene bcl-10 translocated withan IgH locus may be overexpressed in such patients. The expression levelof oncogene bcl-10 translocated with an IgH locus may be detected insuch patients using known methods of measuring or detecting geneexpression. Those patients having or suspected of havingmucosa-associated lymphatic tissue (MALT)-type lymphoma in whichoncogene bcl-10 translocated with an IgH locus is overexpressed maybenefit from administration of an effective amount of a bromodomainprotein inhibitor in accordance with the methods disclosed herein.

In one embodiment, the oncogene is c-maf, the Ig locus is an IgH locus,and the cancer is multiple myleloma. For example, patients having orsuspected of having multiple myleloma may exhibit oncogene c-maftranslocated with an IgH locus. In some instances, oncogene c-maftranslocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene c-maf translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients having or suspected of havingmultiple myleloma in which oncogene c-maf translocated with an IgH locusis overexpressed may benefit from administration of an effective amountof a bromodomain protein inhibitor in accordance with the methodsdisclosed herein. In an embodiment, the c-maf oncogene translocation isa [t(14;16)] chromosomal translocation.

In one embodiment, the oncogene is c-myc, the Ig locus is an IgH locus,and the cancer is Burkitt's lymphoma. For example, patients having orsuspected of having Burkitt's lymphoma may exhibit oncogene c-myctranslocated with an IgH locus. In some instances, oncogene c-myctranslocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene c-myc translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients having or suspected of havingBurkitt's lymphoma in which oncogene c-myc translocated with an IgHlocus is overexpressed may benefit from administration of an effectiveamount of a bromodomain protein inhibitor in accordance with the methodsdisclosed herein. In one embodiment, the c-myc oncogene translocation isa [t(14;16)] chromosomal translocation. In one embodiment, the c-myconcogene translocation is a [t(8;22)] chromosomal translocation. In oneembodiment, the c-myc oncogene translocation is a [t(2;8)] chromosomaltranslocation.

In one embodiment, the oncogene is c-myc, the Ig locus is an IgH locus,and the cancer is multiple myeloma. For example, patients having orsuspected of having multiple myeloma may exhibit oncogene c-myctranslocated with an IgH locus. In some instances, oncogene c-myctranslocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene c-myc translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients having or suspected of havingmultiple myeloma in which oncogene c-myc translocated with an IgH locusis overexpressed may benefit from administration of an effective amountof a bromodomain protein inhibitor in accordance with the methodsdisclosed herein.

In one embodiment, the oncogene is FGFR3, the Ig locus is an IgH locus,and the cancer is multiple myeloma. For example, patients having orsuspected of having multiple myeloma may exhibit oncogene FGFR3translocated with an IgH locus. In some instances, oncogene FGFR3translocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene FGFR3 translocated with an IgH locusmay be detected in such patients using known methods of measuring ordetecting gene expression. Those patients having or suspected of havingmultiple myeloma in which oncogene FGFR3 translocated with an IgH locusis overexpressed may benefit from administration of an effective amountof a bromodomain protein inhibitor in accordance with the methodsdisclosed herein. In one embodiment, the FGFR3 oncogene translocation isa [t(4;14)] chromosomal translocation.

In one embodiment, the oncogene is Lyt-10, the Ig locus is an IgH locus,and the cancer is diffuse large cell lymphoma. For example, patientshaving or suspected of having diffuse large cell lymphoma may exhibitoncogene Lyt-10 translocated with an IgH locus. In some instances,oncogene Lyt-10 translocated with an IgH locus may be overexpressed insuch patients. The expression level of oncogene Lyt-10 translocated withan IgH locus may be detected in such patients using known methods ofmeasuring or detecting gene expression. Those patients having orsuspected of having diffuse large cell lymphoma in which oncogene Lyt-10translocated with an IgH locus is overexpressed may benefit fromadministration of an effective amount of bromodomain protein inhibitorin accordance with the methods disclosed herein. In an embodiment, theLyt-10 oncogene translocation is a [t(10;14)] chromosomal translocation.

In one embodiment, the oncogene is MUC1, the Ig locus is an IgH locus,and the cancer is extranodal lymphoma. For example, patients having orsuspected of having extranodal lymphoma may exhibit oncogene MUC1translocated with an IgH locus. In some instances, oncogene MUC1translocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene MUC1 translocated with an IgH locus maybe detected in such patients using known methods of measuring ordetecting gene expression. Those patients having or suspected of havingextranodal lymphoma in which oncogene MUC1 translocated with an IgHlocus is overexpressed may benefit from administration of an effectiveamount of a bromodomain protein inhibitor in accordance with the methodsdisclosed herein. In an embodiment, the MUC1 oncogene translocation is a[t(1;14)] chromosomal translocation.

In one embodiment, the oncogene is MUM1/IRF4, the Ig locus is an IgHlocus, and the cancer is multiple myeloma. For example, patients havingor suspected of having multiple myeloma may exhibit oncogene MUM1/IRF4translocated with an IgH locus. In some instances, oncogene MUM1/IRF4translocated with an IgH locus may be overexpressed in such patients.The expression level of oncogene MUM1/IRF4 translocated with an IgHlocus may be detected in such patients using known methods of measuringor detecting gene expression. Those patients having or suspected ofhaving multiple myeloma in which oncogene MUM1/IRF4 translocated withIgH locus is overexpressed may benefit from administration of aneffective amount of a bromodomain protein inhibitor in accordance withthe methods disclosed herein. In an embodiment, the MUM1/IRF4 oncogenetranslocation is a [t(6;14)] chromosomal translocation.

In one embodiment, the oncogene is Pax-5, the Ig locus is an IgH locus,and the cancer is lymphoplasmacytoid lymphoma. For example, patientshaving or suspected of having lymphoplasmacytoid lymphoma may exhibitoncogene Pax-5 translocated with an IgH locus. In some instances,oncogene Pax-5 translocated with an IgH locus may be overexpressed insuch patients. The expression level of oncogene Pax-5 translocated withan IgH locus may be detected in such patients using known methods ofmeasuring or detecting gene expression. Those patients having orsuspected of having lymphoplasmacytoid lymphoma in which oncogene Pax-5translocated with IgH locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain protein inhibitorin accordance with the methods disclosed herein. In an embodiment, thePax-5 oncogene translocation is a [t(9;14)] chromosomal translocation.

In one embodiment, the oncogene is Bc16, the Ig locus is an IgL locus,and the cancer is a B-cell non-Hodgkin lymphoma (e.g., diffuse largecell lymphoma, adult aggressive lymphoma, etc). For example, patientshaving or suspected of having a B-cell non-Hodgkin lymphoma may exhibitoncogene Bc16 translocated with an IgL locus. In some instances,oncogene Bc16 translocated with an IgL locus may be overexpressed insuch patients. The expression level of oncogene Bc16 translocated withan IgL locus may be detected in such patients using known methods ofmeasuring or detecting gene expression. Those patients having orsuspected of having a B-cell non-Hodgkin lymphoma in which oncogene Bc16translocated with an IgL locus is overexpressed may benefit fromadministration of an effective amount of a bromodomain protein inhibitorin accordance with the methods disclosed herein. In an embodiment, theBc16 oncogene translocation is a [t(3;22)(q27;q11)] chromosomaltranslocation.

In one embodiment, the oncogene is c-Myc, the Ig locus is an IgL locus,and the cancer is a B-cell lymphoblastic leukemia and/or non-Hodgkinlymphomas (e.g., diffuse large cell lymphoma, adult aggressive lymphoma,Burkitt's lymphoma, etc). For example, patients having or suspected ofhaving a B-cell lymphoblastic leukemia and/or a non-Hodgkin lymphoma mayexhibit oncogene c-Myc translocated with an IgL locus. In someinstances, oncogene c-Myc translocated with an IgL locus may beoverexpressed in such patients. The expression level of oncogene c-Myctranslocated with an IgL locus may be detected in such patients usingknown methods of measuring or detecting gene expression. Those patientshaving or suspected of having B-cell lymphoblastic leukemia and/ornon-Hodgkin lymphomas in which oncogene c-Myc translocated with an IgLlocus is overexpressed may benefit from administration of a bromodomainprotein inhibitor in accordance with the methods disclosed herein. In anembodiment, the c-Myc oncogene translocation is a [t(8:22)(q24;q11)]chromosomal translocation.

The present invention contemplates that the bromodomain proteininhibitor can be an inhibitor of a BET bromodomain protein. In someembodiments, the bromodomain inhibitor is an inhibitor of Brd4 protein.In some embodiments, the bromodomain inhibitor is an inhibitor of Brd2protein. In some embodiments, the bromodomain inhibitor is an inhibitorof Brd3 protein. In an embodiment, the bromodomain inhibitor is aninhibitor of BrdT protein.

It should be appreciated by those skilled in the art that thebromodomain protein inhibitors contemplated by the present invention canbe any type of inhibitor. Examples of suitable inhibitors known to thoseskilled in the art include an antisense oligonucleotide, an aptamer, anintrabody, an oligopeptide, a ribozyme, an siRNA, a shRNA, and a smallmolecule inhibitor of bromodomain proteins.

In one embodiment, the bromodomain protein inhibitor of the presentinvention is a small molecule known as JQ1. JQ1 has selectivity withinthe BET family of bromodomain proteins, and may have selectivity beyondthe family as well. Examples of bromodomains in which JQ1 mayeffectively inhibit include Brd2 (N-terminal), Brd2 (C-terminal), Brd3(N-terminal), Brd3 (C-terminal), Brd4 (N-terminal), Brd4 (C-terminal),and CREBBP. JQ1 is[(S)-4-(4-Chloro-phenyl)-2,3,9-trimethyl-6H-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-aceticacid tert-butyl ester. JQ1 has the structure depicted in Formula Ibelow:

In certain embodiments, the inhibitors of the present invention may beadministered as part of a pharmaceutical composition comprising thebromodomain protein inhibitor alone, the bromodomain inhibitor incombination with a cancer therapeutic agent, the bromodomain proteininhibitor conjugated to a cancer therapeutic agent, and/or thebromodomain protein inhibitor component linked to a delivery vehicle,which are each suitable for downregulating the expression of an oncogenetranslocated with an Ig locus, inhibiting the interaction between abromodomain protein and an Ig regulatory element, and/or treating acancer in which expression of an oncogene translocated with an Ig locusis increased (e.g., a hematological malignancy). Therapeuticformulations comprising the bromodomain proteins can be prepared forstorage according to methods known to those skilled in the art.(REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed. 1980), which is herebyincorporated by reference in its entirety). In some embodiments, thebromodomain inhibitor is administered with a pharmaceutically acceptablecarrier.

The active therapeutic ingredients of the pharmaceutical compositions(i.e. bromodomain inhibitors alone or linked to a cancer therapeuticagent) can be entrapped in microcapsules prepared using coacervationtechniques or by interfacial polymerization, e.g.,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (e.g., liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed. 1980),which is hereby incorporated by reference in its entirety. In someembodiments, the bromodomain inhibitors of the present invention can beconjugated to the microcapsule delivery vehicle to target the deliveryof the therapeutic agent to the site of the tumor. Sustained-releasepreparations may be prepared according to well known methods.

In another embodiment, the therapeutic treatment methods of the presentinvention involve the combined administration of one or more bromodomaininhibitors, in combination with a cancer therapeutic agent, orconjugated to a distinct chemotherapeutic agent, radiotherapeutic agent,or immunotherapeutic agent, resulting in the administration of acocktail of chemotherapeutic, radiotherapeutic, and/or immunotherapeuticagents. In yet another embodiment, the bromodomain inhibitors alone orconjugated to the cancer therapeutic can be administered with one ormore additional chemotherapeutic agents. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in CHEMOTHERAPY SERVICE (M. C. Perry ed., 1992), which ishereby incorporated by reference in its entirety.

In an embodiment, the present invention contemplates the use of abromodomain inhibitor for the treatment of a cancer characterized byoverexpression of an oncogene translocated with an Ig locus.

In an embodiment, the present invention contemplates the use of abromodomain inhibitor for the treatment of a cancer characterized byoverexpression of an oncogene translocated with an Ig locus, with theproviso that said oncogene is not c-Myc.

In an embodiment, the present invention contemplates the use of abromodomain inhibitor for the treatment of a cancer characterized byoverexpression of an oncogene translocated with an Ig locus, with theproviso that said cancer is not AML.

In another aspect, the present invention includes a method foridentifying an agent that interferes with binding of a bromodomainprotein to an Ig regulatory element, such method comprising: (a)contacting a suitably conditioned cell containing a target gene undercontrol of one or more Ig regulatory elements and a bromodomain proteinwhich binds to said one or more Ig regulatory elements and activatesexpression of said target gene with a candidate agent; and (b) detectingexpression of said target gene, wherein decreased expression of saidtarget gene in the presence of said candidate agent as compared withexpression of said target gene in the absence of said candidate agent isindicative of said agent's ability to interfere with binding of saidbromodomain protein to said Ig regulatory element. In some embodiments,the method of identifying an agent that interferes with the binding of abromodomain protein to an Ig regulatory element further comprisescontacting said bromodomain protein immobilized onto a solid supportwith said candidate agent and a known inhibitor of said bromodomainprotein, wherein said candidate agent's ability to outcompete said knowninhibitor for binding to said bromodomain protein is indicative of saidcandidate agent's ability to interfere with binding of said bromodomainprotein to said Ig regulatory element. Competitive binding assays andmethod of immobilizing a substrate to a solid support are routinemethods known to those skilled in the art and any such methods nowknown, or later developed, can be used to assess the ability of thecandidate agent to outcompete a known inhibitor of bromodomain proteinfor binding to the bromodomain. In some embodiments, the method ofidentifying an agent that interferes with the binding of a bromodomainprotein to an Ig regulatory element further comprises comparing a levelof bromodomain protein occupancy at said Ig regulatory element in thepresence of said candidate agent to a control, wherein decreasedbromodomain protein occupancy at said Ig regulatory element in thepresence of said candidate agent as compared to in the absence of saidcandidate agent is indicative of bromodomain inhibitor activity of saidcandidate agent. In some embodiments, the control is the bromodomainprotein occupancy at an Ig regulatory element in the presence of a knownbromodomain inhibitor (e.g., JQ1). Those skilled in the art willappreciate, however, that any known bromodomain inhibitor can be used,including bromodomain inhibitors discovered according to the methodsdescribed herein.

In one embodiment, the method of identifying an agent that interfereswith the binding of a bromodomain protein to an Ig regulatory elementfurther comprises (c) contacting said bromodomain protein immobilizedonto a solid support with said candidate agent and a known inhibitor ofsaid bromodomain protein, wherein said candidate agent's ability tooutcompete said known inhibitor for binding to said bromodomain proteinis indicative of said candidate agent's ability to interfere withbinding of said bromodomain protein to said Ig regulatory element; and(d) comparing a level of bromodomain protein occupancy at said Igregulatory element in the presence of said candidate agent which is ableto outcompete said known inhibitor to a control, wherein decreasedbromodomain protein occupancy at said Ig regulatory element in thepresence of said candidate agent as compared to in the absence of saidcandidate agent is indicative of bromodomain inhibitor activity of saidcandidate agent.

In certain embodiments, the candidate agent interferes withacetyl-lysine recognition by a central hydrophobic cavity of saidbromodomain protein or interferes with acetyl-lysine anchoring by ahydrogen bond of an asparagine residue of said bromodomain protein.

In some embodiments, the agent or candidate agent is selected from thegroup consisting of an antisense oligonucleotide, an aptamer, anintrabody, an oligopeptide, a ribozyme, an siRNA, a shRNA, and a smallmolecule.

In an embodiment, the Ig regulatory element is an IgH regulatoryelement.

In some embodiments, the target gene is a reporter gene (e.g., a genethat encodes for a product that is readily quantifiable, e.g., GFP,etc.). In such instances, expression of the reporter gene can bedetected by routine methods known to those skilled in the art todetermine whether the candidate agent decreases expression of thereporter gene. In such instances, decreased expression of the reportergene is indicative that the candidate agent interfered with binding tothe Ig regulatory element, and that the candidate agent may be a leadcandidate agent for additional screening.

In some embodiments, the target gene is fused to a protein tag. Itshould be appreciated by those skilled in the art that the protein tagmay be used for a variety of purposes toward the characterization thetarget gene and its expression, including purification (e.g., affinitytag (GST)), separation (e.g., chromatography tag (FLAG-tag),visualization (e.g., fluorescence tag (GFP), subsequent analysis (e.g.,epitope tags (HA-tag).

In some embodiments, the protein tag is selected from the groupconsisting of a fluorescent peptide and a poly His tag.

In an embodiment, target gene is an oncogene which is translocated withan Ig locus. In such instance, the target gene may be fused to anothertarget gene as described above for measurement of expression of thetarget gene. In one embodiment, the oncogene is not c-Myc.

In one embodiment, the the bromodomain protein is Brd4, the oncogene isc-Myc, and the Ig regulatory element is an IgH enhancer. In suchembodiment, a method for identifying an agent that interferes withbinding of a Brd4 to an IgH regulatory element comprises: (a) contactinga suitably conditioned cell containing a target gene under control ofone or more IgH regulatory elements (e.g., an IgH enhancer or sequenceadjacent to an enhancer) and a Brd4 protein which binds to said one ormore IgH regulatory elements and activates expression of said targetgene with a candidate agent; and (b) detecting expression of said targetgene, wherein decreased expression of said target gene in the presenceof said candidate agent as compared with expression of said target genein the absence of said candidate agent is indicative of said agent'sability to interfere with binding of said Brd4 protein to said IgHregulatory element. The candidate agents identified by performing steps(a) and (b) can optionally be further screened for their ability tointerfere with binding of Brd4 to an IgH regulatory element byperforming step (c) contacting the bromodomain protein (e.g., Brd4)immobilized onto a solid support with said candidate agent and a knowninhibitor of said bromodomain protein (e.g., JQ1), wherein saidcandidate agent's ability to outcompete said known inhibitor for bindingto said bromodomain protein is indicative of said candidate agent'sability to interfere with binding of said bromodomain protein to said Igregulatory element. The candidate agents identified by performing thescreen described in step (c) can further be screened for its ability tointerfere with binding of the bromodomain protein and the Ig regulatoryelement by performing the step of (d) comparing a level of bromodomainprotein (e.g., Brd 4) occupancy at said Ig regulatory element in thepresence of said candidate agent which is able to outcompete said knowninhibitor (e.g., JQ1) to a control, wherein decreased bromodomainprotein occupancy at said Ig regulatory element (e.g., an IgH enhancer)in the presence of said candidate agent as compared to in the absence ofsaid candidate agent is indicative of bromodomain inhibitor activity ofsaid candidate agent.

In another aspect, a method for identifying an inhibitor of abromodomain protein is disclosed, such method comprising: (a) contactinga cell line expressing an oncogene translocated with an Ig locus with acandidate agent; (b) measuring the level of bromodomain proteinoccupancy at a regulatory element of said Ig locus; and (c) comparingsaid level of bromodomain occupancy at said regulatory element of saidIg locus to a control, wherein decreased bromodomain protein occupancyat said regulatory element of said Ig locus in the presence of thecandidate agent as compared to in the absence of said candidate agent isindicative of bromodomain inhibitory activity of said candidate agent.Those skilled in the art will appreciate that an exemplary method ofperforming such method for identifying an inhibitor of bromodomainprotein is described in further detail in Example 1 below.

Generally, any control which is able to assess the ability of abromodomain inhibitor to interfere with binding of a bromodomain proteinto an Ig regulatory element can be employed in accordance with themethods disclosed herein. In certain embodiments, the control is acontrol bromodomain occupancy level or reference or standard level ofbromodomain protein occupancy at an Ig regulatory element. In someembodiments, the control bromodomain occupancy level is determined bycontacting said cell line with a known bromodomain protein inhibitor,and measuring a level of bromodomain occupancy at said regulatoryelement of said Ig locus.

In certain embodiments, the methods for identifying a bromodomaininhibitor contemplate employing a known bromodomain protein inhibitor asa control or for a functional comparator. In an embodiment, the knownbromodomain protein inhibitor is small molecule JQ1.

It should be appreciated that any oncogene which is capable oftranslocation with an Ig locus can be the subject of the identificationmethods of the present invention. In an embodiment, the oncogene isc-myc.

The present invention contemplates that any cell line which is suitablefor expressing an oncogene translocated with an Ig locus can be used inaccordance with the methods disclosed herein. In an embodiment, the cellline is a multiple myeloma cell line.

Generally, the Ig regulatory element can be any regulatory element orsequence associated with an Ig locus which contributes to the expressionof an oncogene translocated with the Ig locus. The skilled artisan willalso recognize that the Ig regulatory element can comprise a bindingsite that is located at any location within the broader Ig regulatoryregion. In certain embodiments, the Ig regulatory element is an IgHenhancer. In some embodiments, the IgH enhancer is selected from thegroup consisting of E1, E2, E3, and E4. IgH enhancers E1, E2, E3, and E4are described in further detail in Example 1 below. In otherembodiments, the Ig regulatory element is a regulatory element locatedwithin a region or adjacent to a region in which the IgH ehancer islocated. In an embodiment, the Ig regulatory element is a regulatoryelement which shares sequence similarity or sequence complementarily toan IgH enhancer. In an embodiment, the regulatory element comprises asequence that spans approximately 100 bp along an Ig regulatory region.

Those skilled in the art will appreciate that a variety of known androutine methods can be used to measure the level of bromodomain proteinoccupancy at a regulatory element of an Ig locus. In some embodiments,said step of measuring said level of bromodomain protein occupancy atsaid regulatory element of said Ig locus is performed using bromodomainprotein ChIP-PCR analysis. In such embodiments, said ChIP-PCR analysisis performed using oligonucleotide primers complementary to at least aportion of a sequence of said regulatory element. In certainembodiments, said step of measuring said level of bromodomain proteinoccupancy at said regulatory element of said Ig locus is performed usingsequencing techniques. In certain embodiments, said step of measuringsaid level of bromodomain protein occupancy at said regulatory elementof said Ig locus is performed using a microarray.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The details of thedescription and the examples herein are representative of certainembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Modifications therein and other uses will occurto those skilled in the art. These modifications are encompassed withinthe spirit of the invention. It will be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention provides all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. It is contemplated that all embodiments described herein areapplicable to all different aspects of the invention where appropriate.It is also contemplated that any of the embodiments or aspects can befreely combined with one or more other such embodiments or aspectswhenever appropriate. Where elements are presented as lists, e.g., inMarkush group or similar format, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. It should be understood that, in general, wherethe invention, or aspects of the invention, is/are referred to ascomprising particular elements, features, etc., certain embodiments ofthe invention or aspects of the invention consist, or consistessentially of, such elements, features, etc. For purposes of simplicitythose embodiments have not in every case been specifically set forth inso many words herein. It should also be understood that any embodimentor aspect of the invention can be explicitly excluded from the claims,regardless of whether the specific exclusion is recited in thespecification. For example, any one or more nucleic acids, polypeptides,cells, species or types of organism, disorders, subjects, orcombinations thereof, can be excluded.

Where the claims or description relate to a composition of matter, e.g.,a nucleic acid, polypeptide, cell, or non-human transgenic animal, it isto be understood that methods of making or using the composition ofmatter according to any of the methods disclosed herein, and methods ofusing the composition of matter for any of the purposes disclosed hereinare aspects of the invention, unless otherwise indicated or unless itwould be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise. Where the claims ordescription relate to a method, e.g., it is to be understood thatmethods of making compositions useful for performing the method, andproducts produced according to the method, are aspects of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodimentswithin a range of 5% of a number or in some embodiments within a rangeof 10% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue). It should be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It should also beunderstood that unless otherwise indicated or evident from the context,any product or composition described herein may be considered“isolated”.

EXAMPLES Example 1: Bromodomain Proteins Bind Ig Enhancers, RegulatingTranslocated Oncogene Expression and Function

Background and Introduction

c-Myc overexpression in cancer uncouples growth-factor stimulation andcell proliferation. c-Myc can be overexpressed through multiplemechanisms in tumor cells, including gene amplification, chromosomaltranslocation and single nucleotide polymorphisms (SNP). The most commonmechanism is through MYC gene amplifications. This was first identifiedin the HL-60 human premyelocytic leukemia cell line, which contains 10to 20 copies of the MYC gene [4, 5]. Some cancers can contain upwards of150 copies of the MYC gene [6]. Chromosomal translocations occur inhematological malignancies, including Burkitt's lymphoma [7, 8]. Thetranslocations typically occur between MYC and the immunoglobulin heavychain (IgH) locus and MYC expression is then controlled by IgHregulatory elements [7-9]. Because IgH is highly expressed in B- andT-cells, this leads to massive overexpression of MYC and uncontrolledproliferation. Additionally, high-risk cancer SNPs associated withcolorectal, breast, bladder, ovarian and prostate cancer at 8q24 havebeen mapped to MYC enhancer elements [10-14]. These regions formchromosome loops with the MYC promoter region to regulate itsexpression. These SNPs may influence MYC expression by altering theaffinity of transcription factors like Tcf4, a terminal component of theWnt/b-catenin signaling pathway, for binding sites in the enhancerregion [14]. Because of c-Myc's role in promoting proliferation,overexpression of this transcription factor contributes to a key aspectof cancer: unregulated proliferation.

Nevertheless, since the historic discovery of MYC as a human oncogenemore than thirty years ago, a therapeutic approach to modulating c-Myctranscription has remained elusive. The absence of a clearligand-binding domain establishes a formidable obstacle toward directlyinhibiting c-Myc function, a challenging feature of many compellingtranscription factor targets in cancer [22]. High-resolution structuresof the c-Myc/Max complex fail to identify a hydrophobic involutioncompatible with the positioning of an organic small molecule [23].

We therefore have undertaken to target c-Myc transcriptional function byanother means, namely the disruption of chromatin-dependent signaltransduction. c-Myc transcription is associated locally and globallywith increases in histone lysine side-chain acetylation [24-26], acovalent modification of chromatin regionally associated withtranscriptional activation [27, 28]. Histone acetylation templates theassembly of higher-ordered transcriptional complexes by the recruitmentof proteins possessing one or more acetyl-lysine binding modules orbromodomains [29, 30]. Members of the bromodomain and extra-terminal(BET) subfamily of human bromodomains (Brd2, Brd3, Brd4 and Brdt)associate with acetylated chromatin and facilitate transcriptionalactivation by increasing the effective molarity of recruitedtranscriptional activators. Notably, Brd4 has been shown to mark selectM/G1 genes in mitotic chromatin as transcriptional memory and directpost-mitotic transcription [31], notably via direct interaction with thepositive transcription elongation factor complex b (P-TEFb) [32]. Thediscovery that c-Myc regulates promoter-proximal pause release of PolII, also through the recruitment of P-TEFb [33], established a rationalefor targeting BET bromodomains to inhibit c-Myc dependent transcription.

Recently, the development and biochemical characterization of a firstpotent, selective small-molecule inhibitor of BET bromodomains, JQ1, wasreported[34]. JQ1 is a thieno-triazolo-1,4-diazepine which displaces BETbromodomains from nuclear chromatin by competitively binding to theacetyl-lysine recognition pocket. In the present study, we leverage thebiochemical and pharmacologic properties of JQ1 as a chemical probe[35], to interrogate the role of BET bromodomains in Myc-dependenttranscription and to explore the putative role of BET bromodomains ascancer dependencies.

Multiple myeloma (MM) represents an ideal model system for thesemechanistic and translational questions given the known role of MYC indisease pathophysiology. MM is an incurable hematologic malignancy,typified by the accumulation of malignant plasma cells harboring diversegenetic lesions [36]. Rearrangement or translocation of MYC is among themost common somatic events in early and late stage MM [37-42], andtranscriptional profiling identifies Myc pathway activation in more than60% of patient-derived MM cells [43]. Experimental support for thecentral role of c-Myc in the pathogenesis of MM is contributed by aninformative, genetically-engineered murine model of MM. Lineage-specificand stochastic Activation-Induced Deaminase (AID)-dependent activationof a conditional MYC transgene in the late stages of B-celldifferentiation establishes genetically-engineered mice with a plasmacell malignancy that shares clinically relevant features of MM [41].Thus, MYC dysregulation represents a largely unifying molecular featureobserved across the otherwise complex genetic pathophysiology of MM,from the pre-malignant monoclonal gammopathy of undeterminedsignificance (MGUS) to the advanced stage of plasma cell leukemia (PCL).

Results and Discussion

To test BET bromodomain inhibitors as a strategy to downregulate thecMyc transcriptional network and the mechanism of downregulation,multiple myeloma cells were treated with JQ1. Treatment with JQ1 induceda potent antiproliferative effect, consistent with loss of cMycfunction. Furthermore, JQ1 treatment caused rapid downregulation of cMycprotein levels and a genome-wide downregulation of cMyc target genes.

MYC translocation with IGH (8;14) is a common form of deregulation inmultiple myeloma. We reasoned that early and sustained JQ1-inducedsuppression of MYC transcription may be mechanistically explained byphysical interaction of BRD4 with regulatory elements influencing MYCexpression. Indeed, avid binding of BRD4 to established IgH enhancerswas observed by chromatin immunoprecipitation (ChIP) in MM.1S cells(FIG. 1A, 1B), which harbor an IgH insertion proximal to the MYCtranscriptional start site (TSS). BRD4 binding was not observed at fivecharacterized enhancer regions adjacent to the MYC gene [13]. JQ1treatment (500 nM) for 24 hours significantly depleted BRD4 binding toIgH enhancers and the TSS, supporting direct regulation of MYCtranscription by BET bromodomains, and a model whereby BRD4 acts as aco-activator of MYC transcription potentially through long-rangeinteractions with distal enhancer elements. This implicates Brd4 as apositive regulator of translocated IGH-MYC expression and a target fordownregulating MYC in these cells.

Our results show that BET domain inhibition is a viable strategy todownregulate the cMyc transcriptional network in multiple myeloma andpotentially in other blood cancers driven by translocated IGH-MYC.Additionally, because Brd4 binds at the IGH regulatory elements and JQ1treatment competes it off these sites, BET bromodomain inhibition couldbe used to downregulate other oncogenes overexpressed throughimmunoglobulin translocations.

The foregoing results establish the use of bromodomain inhibitors (e.g.,BET bromodomain inhibitors) as an effective strategy to downregulateoncogenes overexpressed through translocation with the Ig (e.g., IgH)regulatory elements. The foregoing results provide evidence thatbromodomain inhibitor treatment works for IGH-cMyc translocations inmultiple myeloma because the target of JQ1 (Brd4) binds these regulatoryelements and treatment with JQ1 competes this protein off chromatin. TheIGH-MYC translocation occurs in a number of other blood cancers,including Burkitt's Lymphoma, for example. Translocation betweenoncogenes and IGH is a common occurrence in many B cell malignancies.Translocations involving IGH with other oncogenes are also found,including the oncogenes Bcl2, Ccnd1 [44], c-Maf, Pax5, Pim1 [45], Bc16,Irf4 [46], Il3, Lyt10, Bc13 and Malt1 [47][48, 49].

Experimental Procedures Used in the Examples

Approximately 1×10⁸ MM1S cells were treated with 500 nM JQ1 or DMSO for24 hours and crosslinked with 1.1% formaldehyde (10× crosslink solutioncontains 11% formaldehyde, 50 mM Hepes pH7.3, 100 mM NaCl, 1 mM EDTApH8.0, 0.5 mM EGTA pH8.0) to the growth media followed by two washeswith PBS. Cells were scraped and frozen in liquid nitrogen. Brd4ChIP-PCR analysis was done on these cells following the protocol in Rahlet al. Cell 2010. In brief, 75 ul of Dynal magnetic beads (Sigma) wereblocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 6.25ug of Brd4 antibody (Bethyl Labs, A310-985A, lot A301-985A-1, FIG. 1A orSigma, HPA015055-100, lot A31530, FIG. 1B). Crosslinked cells were lysedwith lysis buffer 1 (50 mM Hepes pH7.3, 140 mM NaCl, 1 mM EDTA, 10%glycerol, 0.5% NP-40, and 0.25% Triton X-100) and washed with lysisbuffer 2 (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1 mM EDTA pH8.0 and 0.5 mMEGTA pH8.0). Cells were resuspended and sonicated in lysis buffer 3 (50mM Tris-HCl pH7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,0.1% Na-deoxycholate, 0.1% SDS) for 10 cycles at 30 seconds each on ice(18 watts) with 60 seconds on ice between cycles. Sonicated lysates werecleared and incubated overnight at 4° C. with magnetic beads bound withantibody to enrich for DNA fragments bound by the indicated factor.Beads were washed three times with sonication buffer, one time withsonication buffer with 500 mM NaCl, one time with LiCl wash buffer (20mM Tris pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate)and one time with TE. DNA was eluted in elution buffer. Cross-links werereversed overnight. RNA and protein were digested using RNAse A andProteinase K, respectively and DNA was purified with phenol chloroformextraction and ethanol precipitation.

Brd4 ChIP and input DNA were analyzed using SYBR Green real-time PCRanalysis (Applied Biosystems). ENCODE H3K27Ac ChIP-seq data available onthe UCSC genome browser (http://genome.ucsc.edu/ENCODE/) was used toidentify potential tissue-specific MYC enhancer and IGH enhancerregulatory elements and oligos were designed for these regions. Foldenrichment was determined from triplicate PCR reactions at fivepotential enhancer regions adjacent to the MYC gene in non-translocatedcells (MYC_E1, MYC_E2, MYC_E3, MYC_E4, MYC_E5), the MYC transcriptionalstart site (MYC_TS1, MYC_TS2), IGH enhancer regions (IGH_E1, IGH_E2,IGH_E3, IGH_E4), and two negative regions upstream of the MYC enhancers(MYC_NR2, MYC_NR3) over input DNA using DDCt over the negative regionMYC_NR1. The oligos used for this analysis are:

Primer HG19 Region Set Location F/R Sequence SEQ ID NO MYC NR1chr8: 127, 714, 271- FWD GCAGCTAGATCGTTGGGAAG SEQ ID NO: 1 127, 788, 621REV GCTGGTGATTTCAGTGCAGA SEQ ID NO: 2 MYC NR2 chr8: 127, 714, 271-FWD TCCTGGGTAGGAACCAGTTG SEQ ID NO: 3 127, 788, 621REV ACTCACCAAGAGCTCCTCCA SEQ ID NO: 4 MYC NR3 chr8: 127, 714, 271-FWD AAGCCAACCCATCACTGAAC SEQ ID NO: 5 127, 788, 621REV TTCCCATGTTACCCACCACT SEQ ID NO: 6 MYC_E1 chr8: 127, 845, 547-FWD TGCTAATTGTGCCTCTCCTGT SEQ ID NO: 7 127, 846, 357REV ACTCCCAGCAAATCAGCCTA SEQ ID NO: 8 MYC_E2 chr8: 128, 218, 650-FWD TTGTCTTCCTCATGCCTTCTC SEQ ID NO: 9 128, 218, 982REV AGCTGCAATTCAGGGTTACTG SEQ ID NO: 10 MYC_E3 chr8: 128, 237, 690-FWD TTTGGGCAGACACTGACTTG SEQ ID NO: 11 128, 237, 903REV AAGCAGAAACAGGACCCAGA SEQ ID NO: 12 MYC_E4 chr8: 128, 307, 286-FWD GAAATGTGAGGGCACATCGT SEQ ID NO: 13 128, 307, 531REV ATACCTGCTGGAGCATTTGG SEQ ID NO: 14 MYC_E5 chr8: 128, 568, 909-FWD TCAGCCTGTGGGCTCTAGTT SEQ ID NO: 15 128, 569, 272REV AGGGAAGTGCTACCCCATCT SEQ ID NO: 16 MYC_TS1 chr8: 128, 747, 740-FWD ACACTAACATCCCACGCTCTG SEQ ID NO: 17 128, 748, 029REV GATCAAGAGTCCCAGGGAGA SEQ ID NO: 18 MYC_TS2 chr8: 128, 749, 029-FWD GGTCGGACATTCCTGCTTTA SEQ ID NO: 19 128, 749, 395REV GATATGCGGTCCCTACTCCA SEQ ID NO: 20 IGH_E1 chr14: 106, 047, 824-FWD TGGGGTACAAGAGGCTTCAG SEQ ID NO: 21 106, 049, 452REV TACAGGAGTGGGGACAGGAA SEQ ID NO: 22 IGH_E2 chr14: 106, 047, 824-FWD GTTCTCTGGCTGGAACACCT SEQ ID NO: 23 106, 049, 452REV CCCCATCACCTGCAGAAATA SEQ ID NO: 24 IGH_E3 chr14: 106, 167, 019-FWD CCACAGGGCTATTTTGGGTA SEQ ID NO: 25 106, 167, 760REV GCCATGCCGTTTGTATTCTC SEQ ID NO: 26 IGH_E4 chr14: 106, 167, 019-FWD GGGAGGCCATGCTGTTTGTATTCT SEQ ID NO: 27 106, 167, 760REV AACAGCAGTTCTCTGGCTGGAACA SEQ ID NO: 28

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1. (canceled)
 2. A method for downregulating expression of an oncogenetranslocated with an Ig locus comprising contacting said locus with aneffective amount of an inhibitor of a bromodomain protein. 3.-4.(canceled)
 5. The method of claim 2 wherein downregulating expression ofsaid oncogene comprises decreasing the level or activity of anexpression product of said oncogene.
 6. (canceled)
 7. The method ofclaim 2 wherein said oncogene is not c-Myc.
 8. The method of claim 2wherein said oncogene is selected from the group consisting of Bcl2,Ccnd1, c-Maf, Pax5, Pim1, Bc16, Irf4, Il3, Lyt10, Bc13, and Malt1. 9.The method of claim 2 wherein said oncogene is c-Myc. 10.-11. (canceled)12. The method of claim 2 wherein said inhibitor interferes with bindingof said bromodomain protein to a transcriptional start site of saidoncogene. 13.-19. (canceled)
 20. The method of claim 2 wherein saidinhibitor is selected from the group consisting of an anti senseoligonucleotide, an aptamer, an intrabody, an oligopeptide, a ribozyme,an siRNA, a shRNA, and a small molecule.
 21. The method of claim 2wherein said bromodomain protein is a BET bromodomain protein.
 22. Themethod of claim 2 wherein said bromodomain protein is selected from thegroup consisting of Brd2, Brd3, Brd4, and BrdT.
 23. (canceled)
 24. Amethod for treating a cancer comprising: (a) determining the level ofexpression of an oncogene translocated with an Ig locus in a sampleobtained from an individual having or suspected of having said cancer,wherein overexpression of said translocated oncogene in said sample incomparison with a control is indicative that said individual is anindividual who would potentially benefit from treatment with aninhibitor of a bromodomain protein; and (b) administering an effectiveamount of said inhibitor to said individual if said translocatedoncogene is overexpressed in said sample.
 25. A method for treating acancer comprising administering an effective amount of an inhibitor of abromodomain protein to an individual exhibiting overexpression of anoncogene translocated with an Ig locus.
 26. (canceled)
 27. The method ofclaim 24 wherein said cancer is a hematological malignancy.
 28. Themethod of claim 27 wherein said hematological malignancy is a leukemia,lymphoma or myeloma selected from the group consisting of acutelymphoblastic lymphoma (ALL), Burkitt's lymphoma, chronic lymphoidleukemia (CLL), diffuse large cell lymphoma, extranodal lymphoma,follicular lymphoma, lymphoplasmacytoid lymphoma, mantle zone lymphoma,monoclonal gammopathy of undetermined significance (MGUS), multiplemyeloma, and mucosa-associated lymphatic tissue (MALT)-type lymphoma.29.-46. (canceled)
 47. The method of claim 24 wherein said inhibitor isselected from the group consisting of an antisense oligonucleotide, anaptamer, an intrabody, an oligopeptide, a ribozyme, an siRNA, a shRNA,and a small molecule.
 48. The method of claim 24 wherein said inhibitoris small molecule JQ1.
 49. The method of claim 24 wherein said inhibitoris administered with a pharmaceutically acceptable carrier.
 50. Themethod of claim 24 wherein said inhibitor is co-administered with atleast one chemotherapeutic agent. 51.-53. (canceled)
 54. A method foridentifying an agent that interferes with binding of a bromodomainprotein to an Ig regulatory element comprising: (a) contacting asuitably conditioned cell containing a target gene under control of oneor more Ig regulatory elements and a bromodomain protein which binds tosaid one or more Ig regulatory elements and activates expression of saidtarget gene with a candidate agent; and (b) detecting expression of saidtarget gene, wherein decreased expression of said target gene in thepresence of said candidate agent as compared with expression of saidtarget gene in the absence of said candidate agent is indicative of saidagent's ability to interfere with binding of said bromodomain protein tosaid Ig regulatory element.
 55. The method of claim 54 furthercomprising contacting said bromodomain protein immobilized onto a solidsupport with said candidate agent and a known inhibitor of saidbromodomain protein, wherein said candidate agent's ability tooutcompete said known inhibitor for binding to said bromodomain proteinis indicative of said candidate agent's ability to interfere withbinding of said bromodomain protein to said Ig regulatory element.56.-66. (canceled)
 67. A method for identifying an inhibitor of abromodomain protein comprising: (a) contacting a cell line expressing anoncogene translocated with an Ig locus with a candidate agent; (b)measuring the level of bromodomain protein occupancy at a regulatoryelement of said Ig locus; and (c) comparing said level of bromodomainoccupancy at said regulatory element of said Ig locus to a control,wherein decreased bromodomain protein occupancy at said regulatoryelement of said Ig locus in the presence of the candidate agent ascompared to in the absence of said candidate agent is indicative ofbromodomain inhibitory activity of said candidate agent. 68.-75.(canceled)
 76. The method of claim 25 wherein said cancer is ahematological malignancy.
 77. The method of claim 76 wherein saidhematological malignancy is a leukemia, lymphoma or myeloma selectedfrom the group consisting of acute lymphoblastic lymphoma (ALL),Burkitt's lymphoma, chronic lymphoid leukemia (CLL), diffuse large celllymphoma, extranodal lymphoma, follicular lymphoma, lymphoplasmacytoidlymphoma, mantle zone lymphoma, monoclonal gammopathy of undeterminedsignificance (MGUS), multiple myeloma, and mucosa-associated lymphatictissue (MALT)-type lymphoma.
 78. The method of claim 25 wherein saidinhibitor is selected from the group consisting of an antisenseoligonucleotide, an aptamer, an intrabody, an oligopeptide, a ribozyme,an siRNA, a shRNA, and a small molecule.
 79. The method of claim 25wherein said inhibitor is small molecule JQ1.
 80. The method of claim 25wherein said inhibitor is administered with a pharmaceuticallyacceptable carrier.
 81. The method of claim 25 wherein said inhibitor isco-administered with at least one chemotherapeutic agent.